Many pathogenic microorganisms have Type IV pili to attach to and colonize eukaryotic host cells for virulent infection. The type IV pili are long filamentous organelles comprised of thousands of copies of the pilin subunit. The long term objective of this work is to understand the molecular assembly mechanism of this virulence factor by solving the x-ray crystal structures of type IV pilins and the pilus biogenesis proteins. This information will ultimately be used to design inhibitors that block assembly, eukaryotic cell binding, and/or signaling by pili, thereby serving as antibiotics. In particular, during this funding cycle crystallization and x-ray structure determination will be done for the pilin subunit from Pseudomonas aeruginosa and the pilus assembly/motility factor PilT from the hyperthermophile Aquifex aeolicus. The subunit structure of pilin is needed to test the hypothesis that the 3-D subunit structure and oligomeric packing of type IV pili is conserved across species. It will also reveal posttranslational modifications. The structure of the pilin subunit will be used to model the subunit contacts in an assembled pilus fiber. Combined, the subunit structure and oligomer model will be the basis for design of hybrid pilin molecules to test hypotheses about which parts of the pilin molecule must interact specifically with each other and with other proteins in the biogenesis machinery. Two main roles of pili, twitching motility and signaling among bacteria and with eukaryotic cells, require the PilT protein. The PilT structure will reveal PilT surfaces likely to interact with other proteins in the biogenesis pathway. Logical site-directed mutants will be made to test the in vivo roles of these surfaces. Critical functional residues, effector binding sites in addition to the expected nucleotide binding pocket, and possible phosphorylation sites will furthermore be identified. Together with available biochemical and genetic data, these structural results will eventually lead to a high resolution model of the molecular mechanisms of pilus biogenesis and function. This understanding will be the foundation for blocking pilus functions to control infection by microbes.